Elderly individuals are particularly susceptible to cardiopulmonary pathology induced by airborne nanoparticulate matter (nPM). Antioxidant enzymes are induced by the transcription factor Nrf2 upon nPM inhalation in young but not older mice. The loss of antioxidant inducibility correlates with age-related elevation of c-Myc and Bach1, both of which inhibit Nrf2-regulation of antioxidant enzyme transcription. In aging, inflammatory cytokine production increases both basally and in response to nPM exposure. We hypothesize that the increase in Nrf2-inhibitory proteins in response to nPM during aging is responsible for suppressing antioxidant enzyme inducibility. We further hypothesize that the decrease in Nrf2-dependent antioxidant response in aging permits a greater nPM-induced, NF-kB-regulated increase in inflammatory cytokines. Primary human bronchial epithelium (NHBE) and M1 and M2 macrophages differentiated from peripheral blood monocytes (PBMC) from individuals in four age ranges will be used to model human nPM exposure. Ambient nPM is a mixture of different particle shapes, sizes, and compositions. Thus, we will manufacture defined, reproducible model nPM with physico-chemical characteristics derived from high-resolution microscopy and spectroscopy of ambient nPM from a region with one of the highest nPM health risks. Aim 1 is to demonstrate that the loss in Nrf2-regulated inducibility of antioxidant enzymes (glutamate cysteine ligase, NAD(P)H:quinone oxidoreductase 1, and heme oxygenase-1) in aging is caused by elevation of Nrf2- inhibitory proteins. Bach1 and/or c-Myc will be silenced or overexpressed in NHBE or M1 or M2 macrophages, and expression of Nrf2-regulated antioxidant enzymes in response to nPM determined. Aim 2 is to demonstrate that the decreased ability to induce Nrf2-regulated antioxidant enzymes in aging significantly contributes to elevated inflammatory cytokine production in response to nPM. We will determine the effects of silencing or overexpressing Nrf2, c-Myc and/or Bach1 on NF-kB activation of pro-inflammatory cytokines in response to nPM in NHBE and M1 cells from donors of different ages. The effects of aging and nPM exposure on TNF alpha-induced anti-inflammatory IL-10 expression in M2 cells will also be examined. Aim 3 is to demonstrate that reduced glutathione diethyl ester (GSH-E) or microRNAs (miRNA) that target Bach1 can reverse the attenuation of inducible antioxidant defense in aging. GSH-E, which is converted to glutathione in cells or Bach1-directed miRNAs will be used in NHBE and M1 and M2 macrophages of different ages. Basal and nPM-induced NF-kB activation and induction of cytokines will be determined. The use of human models and representative, reproducible nPM will provide a new mechanistic framework to resolve links among nPM-induced inflammation, antioxidant defense and aging. Acquiring this new knowledge is critical to achieving the ultimate goal of identifying novel means to reduce nPM-induced oxidative injury and inflammation in susceptible individuals, among who are the increasing US elderly population.